The major goal of the project is to understand how the human translation initiation factor elF3 contributes[unreadable] to the initiation mechanism and to the regulation of viral mRNAs, especially Hepatitis C Virus (HCV) mRNA[unreadable] that initiates translation by ribosome binding to an internal entry site (IRES). The structure of elF3, a factor[unreadable] comprising 12 non-identical subunits, will be probed by isolating a variety of sub-complexes formed in[unreadable] baculovirus-infected insect cells. The binding of elF3 and its sub-complexes to the HCV IRES will be defined[unreadable] quantitatively. A highly active partially fractionated mammalian cell lysate system dependent on purified elF3[unreadable] will be constructed with (5-globin mRNA. This system will be used to define the role of elF3 and its subcomplexes[unreadable] in the translation of HCV mRNA. An in vitro translation system also will be constructed that[unreadable] utilizes all of the purified initiation factors, in order to define which factors are required for the translation of[unreadable] HCV and Dengue virus mRNAs. These studies will determine to what extent the viral mRNAs differ from[unreadable] capped/polyadenylated mRNAs in their initiation mechanisms, and may identify targets for therapeutic[unreadable] intervention of infections by these viruses. Considerable evidence indicates that elF3 plays an important role[unreadable] in regulating protein synthesis and cell proliferation. elF3, like other initiation factors, is phosphorylated on[unreadable] numerous subunit proteins. The sites of phosphorylation will be identified by mass spectroscopy, and the[unreadable] effects of phosphorylation on function will be tested in the translation assays. Mutant forms will be[unreadable] constructed and tested, with phosphorylation sites substituted with alanine to prevent phosphorylation, and[unreadable] with glutamate or aspartate to mimic phosphorylation. The experiments are designed to elucidate the[unreadable] mechanism of action of elF3 and the role of phosphorylation in regulating its activity during initiation of[unreadable] capped and IRES-driven mRNAs. The results may explain how disfunction or disregulation of elF3 may[unreadable] affect the control of cell proliferation, resulting in celt malignancy.